ST3Gal-4 is the primary sialyltransferase regulating the synthesis of E-, P-, and L-selectin ligands on human myeloid leukocytes

Abstract

• A single a(2,3) sialyltransferase, ST3Gal-4, controls sLe X biosynthesis on N-and O-glycans in cells of human myeloid lineage.• Blocking this enzyme activity prevents human neutrophil adhesion to E-, P-, and L-selectin.The precise glycosyltransferase enzymes that mediate selectin-ligand biosynthesis in human leukocytes are unknown. This knowledge is important because selectin-mediated cell tethering and rolling is a critical component of both normal immune response and various vascular disorders. We evaluated the role of 3 a(2,3)sialyltransferases, ST3Gal-3, -4, and -6, which act on the type II N-Acetyllactosamine structure (Galb1,4GlcNAc) to create sialyl Lewis-X (sLe X ) and related sialofucosylated glycans on human leukocytes of myeloid lineage. These genes were either silenced using lentiviral short hairpin RNA (shRNA) or functionally ablated using the clustered regularly interspaced short palindromic repeat/ Cas9 technology. The results show that ST3Gal-4, but not ST3Gal-3 or -6, is the major sialyltransferase regulating the biosynthesis of E-, P-, and L-selectin ligands in humans. Reduction in ST3Gal-4 activity lowered cell-surface HECA-452 epitope expression by 75% to 95%. Glycomics profiling of knockouts demonstrate an almost complete loss of the sLe X epitope on both leukocyte N-and O-glycans. In cell-adhesion studies, ST3Gal-4 knockdown/knockout cells displayed 90% to 100% reduction in tethering and rolling density on all selectins. ST3Gal-4 silencing in neutrophils derived from human CD34 1 hematopoietic stem cells also resulted in 80% to 90% reduction in cell adhesion to all selectins. Overall, a single sialyltransferase regulates selectin-ligand biosynthesis in human leukocytes, unlike mice where multiple enzymes contribute to this function. (Blood. 2015;125(4):687-696)

Multi-level regulation of cellular glycosylation: from genes to transcript to enzyme to structure

“…Data from such experiments, in tandem with genome editing methods (TALEN, CRISPR-Cas9 etc. ), may enable estimation of glycosyltransferase activities and dissection of the interdependencies among glycogenes in the same biosynthetic pathway [5,39]. …”

“…In another study of O-linked glycosylation, Liu et al studied the critical enzymes (fucosyltransferases and sialyltransferases) regulating microheterogeneity in the leukocyte cell surface protein P-selectin glycoprotein ligand-1 and leukocyte cell adhesion function [45]. Predictions from such modeling have been validated by developing RNA-interference and CRISPR-Cas9 based knockouts [39,46]. In another example, Bennun et al .…”

Glycosphingolipids on Human Myeloid Cells Stabilize E-Selectin–Dependent Rolling in the Multistep Leukocyte Adhesion Cascade

“…1A-C could be completely blocked by anti-E-selectin mAbs (HAE-1f or P2H3) and cell binding in Fig. 1D-F was abrogated by anti-P-selectin mAb G1 (data not shown, 7, 25, 26 ).…”

“…Genome editing allows complete deletion of specific enzyme activity unlike RNAi, which is incomplete 25, 28 . HL-60 knockouts were thus generated using CRISPR-Cas9 method (Figure 3).…”

Modulating Cell‐Surface Receptor Signaling and Ion Channel Functions by In Situ Glycan Editing

“…[12] In vivo ST3Gal-IV is known to add sialic acids onto the terminal galactose of N- and O-glycans, [13] however, the in vitro specificity and substrate scope of recombinant human ST3Gal-IV for cell-surface glycan editing has not been characterized. Kinetic analysis using type II LacNAc as the acceptor substrate revealed that this enzyme has excellent activity toward the natural donor substrate CMP-Neu5Ac (CMP-Sia) with K m of 0.0734 mM and V max of 0.0478 nmol min −1 .…”